CN105327367B - Imaging agent, preparation method and application - Google Patents
Imaging agent, preparation method and application Download PDFInfo
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- CN105327367B CN105327367B CN201510789274.8A CN201510789274A CN105327367B CN 105327367 B CN105327367 B CN 105327367B CN 201510789274 A CN201510789274 A CN 201510789274A CN 105327367 B CN105327367 B CN 105327367B
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- 150000003242 quaternary ammonium salts Chemical class 0.000 claims abstract description 15
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- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims abstract description 8
- 229960004657 indocyanine green Drugs 0.000 claims abstract description 8
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 39
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 3
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
- A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/225—Microparticles, microcapsules
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nanotechnology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to an imaging agent, which is characterized by comprising IR820-CSQ-Fe nanoparticles, wherein the IR820-CSQ-Fe nanoparticles are ferroferric oxide coated with the following structure: A-X-B; wherein A is chitosan quaternary ammonium salt CSQ with a structure of formula (I): wherein n is an integer of 17-290; b is a novel indocyanine green IR820 having the structure of formula (II): x is a compound having the structure of formula (III): wherein R is an alkylene group. The imaging agent has superparamagnetism, prominent relaxation enhancement effects of T1 and T2, photoacoustic imaging capability, obvious fluorescence spectrum, small and uniform particle size, good penetrability, no obvious cytotoxicity and good biocompatibility; the long circulation effect is better; the preparation method is simple, mild in condition and low in cost, and can be freeze-dried and stored for a long time in a solid form.
Description
Technical Field
the invention belongs to the field of nano imaging agents, particularly relates to a multi-mode imaging agent, and a preparation method and application thereof, and particularly relates to an imaging agent for relaxation enhancement, fluorescence imaging or photoacoustic imaging of T1 and T2, which can be used for MRI imaging, and particularly relates to an imaging agent which can be used as a tumor-targeted T1-T2 binuclear magnetic resonance imaging contrast agent.
Background
currently, for a serious disease, it is very limited to determine a lesion site or a lesion degree by only one imaging means, and it is a trend that imaging can be performed in a plurality of ways.
the sensitivity of fluorescence imaging is particularly high, but the tissue penetration capability is poor; the photoacoustic imaging has higher sensitivity and stronger penetrating power, but the penetrating power of laser is limited, so that the deep tissue still has certain limitation; the penetrating power of nuclear magnetic imaging is very strong, and spatial resolution is higher, and the quality of can imaging is very big with the state relation of measurand, and slight motion wants to cause motion artifact, will influence the definition of image like this, and then influences the analysis to the state of an illness, causes the erroneous judgement. If the several imaging modes can be combined and analyzed together, the analysis of the disease condition can be more accurate.
Indocyanine green (ICG) is one of the heptamethine cyanine dyes, and since both absorption and emission spectra are in the near infrared region, it can increase the tissue penetration ability of laser light, and is used as an imaging agent for photoacoustic imaging. The novel indocyanine green (IR820) is formed by introducing a bridge ring into an ICG molecular structure, so that although the fluorescence quantum yield is reduced, the stability is improved; at the same time, the chlorine atom on the bridged ring also provides an active site for further chemical modification. At present, IR820 has become the first choice for scientific research.
According to the contrast enhancement type, currently used nuclear magnetic resonance contrast agents are divided into two categories: positive contrast agents and negative contrast agents. They are respectively enhanced by 1/T of local region1Or 1/T2The relaxation efficiency achieves the effect that the image signal enhances the brightness of the picture or the darkening of the picture. The commonly used negative contrast agent is iron, but many of them have unstable properties due to large particle size, or are only stable in organic phase due to the limitation of preparation method, so that the application is limited. In addition, most contrast agents are only availablethere is an effect of contrast enhancement that two contrast effects cannot be simultaneously achieved, so that accuracy in diagnosing a disease is limited.
for example, the nuclear magnetic resonance contrast agent gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA), although approved for clinical use, has limited applications because its chelated gadolinium ions are easily released, causing toxicity, and do not have long-circulating and targeting effects; in addition, this contrast agent has T1And T2simultaneous contrast effect, however T1The contrast effect is better than T2Contrast effect; the contrast effect of the negative contrast agent SPIO is the opposite.
CN104174037A discloses a probe with T1、T2a process for preparing the doped contrast medium with contrast function features that the dual-head amphiphilic organic molecule is used as template agent, the rare-earth ions and VIB-VIII B ions are mixed in the presence of aromatic acid, and the mixture prepared in self-steaming system has nano-class particle diameter and better T1、T2Contrast effect; however, it is not stable in aqueous solution for a long period of time, and is complicated to operate and expensive.
Chitosan is an aminopolysaccharide composed of 2-amino-D-glucose repeats, which is a deacetylated product of chitin. Because of the advantages of low cost, biodegradability, good biocompatibility, easy chemical modification, small secondary pollution and the like, the chitosan is widely applied to the fields of water treatment, membrane technology, medical bioengineering and the like and is one of natural polymers with the most excellent performance. But its poor solubility in water limits its use in many ways. If quaternary ammonium salt side chains are introduced to the chitosan molecule, the number of positive charges can be increased, thereby greatly improving the water solubility thereof. The new chitosan derivative chitosan quaternary ammonium salt (CSQ) overcomes the defect of poor solubility of chitosan, can be well dissolved under physiological conditions, and is obviously superior to chitosan in the aspects of biocompatibility, antibacterial property, moisture absorption, moisture retention and the like, so the application range of the chitosan derivative chitosan quaternary ammonium salt is quite wide. The chitosan quaternary ammonium salt is a potential absorption enhancer of water-soluble drugs due to the excellent mucosa adsorbability and permeability.
Accordingly, there is a need in the art to develop a multimodal imaging agent that can be simultaneously applied to nuclear magnetic imaging, fluorescence imaging, and/or photoacoustic imaging.
Disclosure of Invention
in view of the defects of the prior art, one of the objects of the present invention is to provide an imaging agent, which comprises IR820-CSQ-Fe nanoparticles, wherein the IR820-CSQ-Fe nanoparticles are ferroferric oxide coated with the following structure:
A-X-B；
Wherein A is chitosan quaternary ammonium salt CSQ with a structure of formula (I):
wherein n is an integer of 17-290; n may take the value of 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, etc., for example; if the molecular weight of CSQ is too small, namely the polymerization degree is low, the viscosity of the solution can be reduced, the effect of blocking the growth of ferroferric oxide crystals cannot be achieved, and proper nanoparticles cannot be prepared; if the molecular weight of CSQ is too large, especially if the polymerized unit n is more than 320, the CSQ is unstable in physiological environment due to too strong positive charge, and the viscosity is too large, thereby affecting the contrast effect; the particle size uniformity of the composite magnetic nanoparticles of the present invention may also be affected by the CSQ having too large or too small a molecular weight.
b is a novel indocyanine green IR820 having the structure of formula (II):
x is a compound having the structure of formula (III):
wherein R is an alkylene group.
the term "comprising" as used herein means that in addition to the IR820-CSQ-Fe nanoparticles, an aqueous dispersion system for dispersing the IR820-CSQ-Fe nanoparticles, such as PBS (phosphate buffered saline) or physiological saline, and other additives may be added as required by those skilled in the art.
In the structure of the IR820-CSQ-Fe nano particle, A is chitosan quaternary ammonium salt CSQ with a structure shown in a formula (I), B is novel indocyanine green IR820 with a structure shown in a formula (II), X is a compound with a structure shown in a formula (III), and the connected structure is as follows:
preferably, R is C1～C10linear or branched alkylene of, preferably C1～C10Linear alkylene groups of (1).
illustratively, R may be 1, 3-propylene, 1, 4-butylene, 1, 5-pentylene, 1, 6-hexylene, 1, 7-heptylene, 1, 8-octylene, 1, 9-nonylene, 1, 10-nonylene, 2-methyl-1, 4-butylene, 3-ethyl-1, 6-hexylene, 2-methyl-3-methyl-1, 5-pentylene, and the like.
Preferably, X is
Preferably, the particle size of the IR820-CSQ-Fe nano-particles is 10-30 nm, and exemplary particles can be 10nm, 15nm, 20nm, 25nm or 30nm and the like. If the particle size is larger than 30nm, the T1 imaging effect of the IR820-CSQ-Fe nano-particles is weakened; if the particle size is less than 10nm, the T2 imaging effect of the IR820-CSQ-Fe nanoparticles will be significantly diminished.
preferably, the hydrated particle size of the IR820-CSQ-Fe nanoparticles is 20-100 nm, and may be 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm or 100nm, for example. If the hydrated particle size is larger than 100nm, the T1 imaging effect of the IR820-CSQ-Fe nanoparticles will be reduced; if the hydrated particle size is less than 10nm, the T2 imaging effect of the IR820-CSQ-Fe nanoparticles will be significantly diminished.
preferably, the mass ratio of the iron atoms, the IR820 and the CSQ in the IR820-CSQ-Fe nanoparticles is (3.1-5.2): 1-8.1): 100, typically but not limited to 3.1:1:100, 3.1:4:100, 3.1:8.1:100, 4:1:100, 4:4:100, 4:8.1:100, 5.2:1:100, 5.2:4:100, 5.2:8.1:100 and the like.
it is a second object of the present invention to provide a process for the preparation of an imaging agent according to the first object, said process comprising the steps of:
(1) preparation of CSQ-Fe:
dissolving CSQ in deionized water, continuously introducing protective gas, heating, and preserving heat to obtain a reaction system A;
Dissolving trivalent ferric salt and divalent ferric salt in strong acid solution to obtain solution B;
thirdly, injecting the solution B into the reaction system A, adjusting the pH value to be alkaline, and refluxing to obtain a reaction system C;
Cooling the reaction system C to room temperature, and dialyzing to obtain a colloid D, namely CSQ-Fe;
(2) preparation of IR820-CSQ-Fe nanoparticles
IR820 andDissolving in an organic solvent, continuously introducing inert gas, heating, and keeping the temperature to obtain a reaction system E; r is alkylene;
Adding organic amine into the reaction system E, stirring and reacting in the dark, and cooling to room temperature to obtain a reaction system F;
③ will andContacting equimolar carbodiimide condensing agent and acylation activating agent with the reaction system F to obtain an activated IR820 solution;
Dripping the activated IR820 solution into the colloid D, and reacting at room temperature in a dark place;
Dialyzing and freeze-drying to obtain the imaging agent containing the IR820-CSQ-Fe nano particles, and storing at 4 ℃ in a dark place.
in the step (2), the acylation activator may be contacted with the reaction system F in an exemplary manner by adding the acylation activator to the reaction system F.
Preferably, the mass ratio of the iron element, the IR820 and the chitosan quaternary ammonium salt CSQ is (3.1-5.2): (1-8.1): 100, typical but not limiting examples of the mass ratio of iron element, IR820, and chitosan quaternary ammonium salt CSQ may be 3.1:1:100, 3.1:4:100, 3.1:8.1:100, 4:1:100, 4:4:100, 4:8.1:100, 5.2:1:100, 5.2:4:100, 5.2:8.1:100, and the like. The mass ratio of the three components is limited in order to ensure that a proper amount of CSQ with proper molecular weight is combined on the surface of the ferroferric oxide nano-particle, and simultaneously ensure the fluorescence property of IR820 to form the composite magnetic nano-particle with uniform and proper particle size; and the weakening of T1 imaging effect or T2 imaging effect caused by too large or too small molecular weight of CSQ or ferroferric oxide particle size or too low fluorescence efficiency caused by fluorescence quenching and too small of IR820 caused by too much amount is avoided.
Preferably, the concentration of CSQ in the reaction system A in the step (1) is 30-60 g/L, and typically but not limited to 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L or 60 g/L. The concentration of the CSQ in the reaction system A is the amount of the CSQ added during the reaction, and the excessive or insufficient concentration can cause the particle size of the finally formed composite magnetic nanoparticles to be too large or too small, thereby affecting the imaging effect of T1 or the imaging effect of T2.
Preferably, in step (1), the protective gas is preferably any 1 or a combination of at least 2 of nitrogen and/or an inert gas, and further preferably nitrogen and/or argon. The purpose of the inert gas is to protect the hyaluronic acid from oxidation, so that the subsequent reaction can be carried out.
Preferably, the temperature for the incubation in step (1) is preferably 80 to 120 ℃, and may be, for example, 80 ℃, 82 ℃, 84 ℃, 86 ℃, 88 ℃, 90 ℃, 92 ℃, 94 ℃, 96 ℃, 98 ℃, 100 ℃, 102 ℃, 104 ℃, 106 ℃, 108 ℃, 110 ℃, 112 ℃, 114 ℃, 116 ℃, 118 ℃ or 120 ℃. The ferroferric oxide nano-particles with uniform particle size can be obtained within a proper temperature range, otherwise, the particle size is not uniform, and the imaging effect is not good.
Preferably, in the step (1), the heat preservation time is preferably 20-50 min, and may be, typically but not limited to, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min or 50 min.
Preferably, the temperature rise in the step (1) is oil bath temperature rise so as to ensure that the liquid which is heated uniformly and is heated within the range of 80-120 ℃ is not evaporated.
preferably, the molar ratio of the ferric iron salt to the ferrous iron salt in the step (1) is 2 (1-2), and typically but not limited to, 2:1, 2:1.1, 2:1.2, 2:1.3, 2:1.4, 2:1.5, 2:1.6, 2:1.7, 2:1.8, 2:1.9 or 2:2, etc. According to the composition ratio of ferric iron to ferrous iron in the ferroferric oxide nano particles, the molar ratio of ferric iron salt to ferrous iron salt is 2:1, and the molar ratio of ferrous iron to ferric iron salt is limited to 2: (1-2) to compensate for the loss of oxidized ferrous iron.
Preferably, the trivalent iron salt in step (1) is FeCl3·6H2O, the ferrous salt is preferably FeCl2·4H2O or FeSO4·7H2O。
Preferably, in the strong acid solution in the step (1), the concentration of the strong acid is 0.5-3 moL/L, and typically but not limited to 0.5moL/L, 0.6moL/L, 0.7moL/L, 0.8moL/L, 0.9moL/L, 1moL/L, 1.1moL/L, 1.2moL/L, 1.5moL/L, 1.8moL/L, 2moL/L, 2.5moL/L, or 3moL/L, etc., and for the added strong acid, hydrochloric acid or nitric acid, etc. may be used to ensure that the reaction does not generate precipitate; if weak acid is added, precipitation can be generated, and the preparation of products is influenced.
Preferably, in step (1), the concentration of ferric ions in the strong acid solution is 0.4-1 g/L, such as 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, etc., and the concentration of ferrous ions in the strong acid solution is 0.2-1 g/L, such as 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, etc.
Preferably, the reagent for adjusting the pH in step (1) is an alkali, preferably ammonia, more preferably ammonia with a mass fraction of 25-28%, typically but not limited to 25%, 25.1%, 25.2%, 25.3%, 25.4%, 25.5%, 25.6%, 25.7%, 25.8%, 25.9%, 26%, 26.1%, 26.2%, 26.3%, 26.4%, 26.5%, 26.6%, 26.7%, 26.8%, 26.9%, 27%, 27.1%, 27.2%, 27.3%, 27.4%, 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, or 28%, and the like, and the pH adjustment to the alkali preferably adjusts the pH to 8.11, and exemplary pH may be 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9.9, 9.5, 9.9, 9.5, 9.9, 9.9.9, 9.9, 9.5, 9.9, 9, 9.5, 9.
preferably, the refluxing time in the step (1) is 40-120 min, for example, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min, 100min, 105min, 110min, 115min or 120 min; the temperature of the reflux is 80-120 ℃, for example, 80 ℃, 82 ℃, 84 ℃, 86 ℃, 88 ℃, 90 ℃, 92 ℃, 94 ℃, 96 ℃, 98 ℃, 100 ℃, 102 ℃, 104 ℃, 106 ℃, 108 ℃, 110 ℃, 112 ℃, 114 ℃, 116 ℃, 118 ℃ or 120 ℃ and the like. The time and temperature of the high-temperature reflux are limited to ensure that IR820-CSQ-Fe nano particles with certain particle sizes are formed, and if the time of the reflux is too short and/or the temperature is too low, crystals are difficult to form; if the refluxing time is too long and/or the temperature is too high, the particle size of the formed IR820-CSQ-Fe nanoparticles is too large, which affects the imaging effect of T1.
Preferably, the dialysis in the step (1) is dialysis with deionized water; the dialysis temperature is preferably 20-35 ℃, for example, can be 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃ etc.; the dialysis time is preferably 60 to 75 hours, and may be, for example, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, or 75 hours.
Preferably, the step of(2) in (1) theIn which R is C1～C10Linear or branched alkylene of, preferably C1～C10Linear alkylene groups of (1).
Illustratively, R may be 1, 3-propylene, 1, 4-butylene, 1, 5-pentylene, 1, 6-hexylene, 1, 7-heptylene, 1, 8-octylene, 1, 9-nonylene, 1, 10-nonylene, 2-methyl-1, 4-butylene, 3-ethyl-1, 6-hexylene, 2-methyl-3-methyl-1, 5-pentylene, and the like.
Preferably, theis 6-aminocaproic acid.
preferably, in step (2) (. sup.R) said IR820 andThe molar ratio of (a) to (b) is 1 (5 to 10), and for example, may be 1: 5. 1: 6. 1: 7. 1:8.1: 9. 1: 10.
Preferably, in step (2), the organic solvent is selected from N, N-Dimethylformamide (DMF) and/or dimethyl sulfoxide (DMSO); the dosage of the organic solvent is 1-10 mL per 15mg of IR 820; for example, the amount of the organic solvent used per 15mg of IR820 may be 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, or the like.
Preferably, in step (2), the protective gas is preferably any 1 or a combination of at least 2 of nitrogen and/or inert gas, further preferably nitrogen and/or argon; the temperature of the heat preservation is preferably 80 to 90 ℃, such as 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, and the like, preferably 85 ℃; the heat preservation time is preferably 20-50 min, and can be, typically but not limited to, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min or 49 min.
Preferably, the temperature rise in the step (2) is oil bath temperature rise to ensure that the liquid which is heated uniformly and is heated within the range of 80-120 ℃ is not evaporated.
preferably, step (2) the organic amine is selected from Triethylamine (TEA) and/or N, N-Diisopropylethylamine (DIEA); in the reaction system F, organic amine andThe molar ratio of (1-5) to (1) may be, for example, 1: 1. 2:1. 3: 1. 4: 1. 5: 1, etc.
preferably, the stirring reaction time in the step (2) is 2-4 h, such as 2.3h, 2.7h, 3.3h, 3.6h, 3.9h and the like, and preferably 3 h.
preferably, in the step (2), the carbodiimide condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and/or N, N-Dicyclohexylcarbodiimide (DCC); the acylation activating agent is preferably N-hydroxysuccinimide (NHS) and/or 1-hydroxybenzotriazole (HOBt);
Preferably, the molar ratio of the IR820 to the CSQ in the colloid D in the step (2) is 1: 20-200, and can be 1:25, 1:30, 1:50, 1:70, 1:100, 1:150, 1:180 and the like; the reaction time is 16-24h, for example, 17h, 18h, 19h, 20h, 21h, 22h, 23 h, etc.
Preferably, the fifth dialysis in the step (2) is dialysis with deionized water; the dialysis temperature is preferably 20-35 ℃, for example, can be 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃ etc.; the dialysis time is preferably 60 to 75 hours, and may be, for example, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, or 75 hours.
As a preferred technical scheme, the preparation method of the imaging agent comprises the following steps:
(1) preparation of CSQ-Fe
Dissolving CSQ in deionized water, wherein the molecular weight of the CSQ is 10,000-100,000 Da; the concentration of the CSQ aqueous solution is 30-60 g/L, nitrogen or argon is introduced for 20-50 min, and then the CSQ aqueous solution is placed in an oil bath to be heated to 80-120 ℃ to obtain a reaction system A;
Dissolving trivalent ferric salt and divalent ferric salt in strong acid to obtain solution B; the molar ratio of the ferric iron salt to the ferrous iron salt is 2 (1-2); the trivalent ferric salt is FeCl3·6H2o; the ferrous salt is FeCl2·4H2O or FeSO4·7H2o; the concentration of the strong acid solution is 0.5-3 moL/L;
Thirdly, injecting the solution B into the reaction system A, adjusting the pH to 8-11 by using 25-28% ammonia water by mass fraction, and refluxing for 40-120 min at 80-120 ℃ to obtain a reaction system C;
Cooling the reaction system C to room temperature, and dialyzing at 20-35 ℃ for 60-75 h to obtain the colloid D.
(2) Preparation of IR820-CSQ-Fe
Dissolving IR820 and 6-aminocaproic acid in N, N-Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) according to a molar ratio of 1 (5-10), introducing nitrogen or argon for 20-50 min, and heating in an oil bath at 85 ℃ to obtain a reaction system E;
adding Triethylamine (TEA) or N, N-Diisopropylethylamine (DIEA) with the molar ratio of (1-5) to 6-aminocaproic acid to the reaction system E, stirring for reaction for 3 hours in a dark place, and cooling to room temperature to obtain a reaction system F;
Contacting 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and/or N, N-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and/or 1-hydroxybenzotriazole (HOBt) which are equimolar with 6-aminocaproic acid with a reaction system F to obtain an activated IR820 solution;
dripping the activated IR820 solution with the molar ratio of the activated IR820 solution to the secondary amine of the CSQ in the colloidal solution being 1: 20-200 into the colloid D, and reacting for 16-24h in a dark place;
And fifthly, dialyzing at 20-35 ℃ for 60-75 h, freezing and drying to obtain the imaging agent, and storing at 4 ℃ in a dark place.
It is a further object of the present invention to provide the use of an imaging agent as defined in one of the objects, which imaging agent is capable of being used in any 1 or at least 2 combination of T1 and T2 relaxation enhancement, fluorescence imaging or photoacoustic imaging for MRI imaging;
Preferably, the imaging agent is used as a tumor-targeted T1-T2 binuclear magnetic resonance imaging contrast agent.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the imaging agents of the invention have superparamagnetic and prominent T1And T2Relaxation enhancing effect, T1and T2the linear relation of the inverse relaxation time along with the change of the iron concentration in a low concentration range is good, the photoacoustic imaging capability is realized, the fluorescence spectrum is obvious, and the method is an ideal imaging agent for preparing the multimode imaging;
(2) the imaging agent has small particle size, good penetrability, uniform particle size and easy purification, and the surface of the particle is positively charged; no obvious cytotoxicity and good biocompatibility; has better long circulation effect, and can be metabolized by the organism after the function is exerted.
(5) the imaging agent of the invention has simple preparation method, mild condition, lower cost and easy popularization and application.
(6) the imaging agent of the invention can be lyophilized and stored in a solid form for a long period of time; when reconstituted with physiological saline, the properties hardly change.
drawings
FIG. 1 is a chart of the infrared spectra of an imaging agent comprising IR820-CSQ-Fe nanoparticles and CSQ-Fe without attachment of IR820 of example 1;
FIG. 2 shows UV absorption spectra of the imaging agent comprising IR820-CSQ-Fe nanoparticles, IR820 and CSQ-Fe of example 1;
FIG. 3 shows a hydrated particle size distribution plot of an imaging agent comprising IR820-CSQ-Fe nanoparticles and CSQ-Fe nanoparticles of example 1;
FIG. 4 shows the surface potential diagram of the imaging agent comprising IR820-CSQ-Fe nanoparticles of example 1;
FIG. 5a shows a transmission electron micrograph of CSQ-Fe nanoparticles of example 1; FIG. 5b shows a transmission electron micrograph of CSQ-Fe nanoparticles of example 5;
FIG. 6 shows an X-ray energy spectrum of the imaging agent of example 1 comprising IR820-CSQ-Fe nanoparticles;
FIG. 7 shows the fluorescence emission spectra of the imaging agent of example 1 comprising IR820-CSQ-Fe nanoparticles at an excitation wavelength of 645 nm;
In FIG. 8, (a) shows the photoacoustic imaging signal intensity of the imaging agent comprising IR820-CSQ-Fe nanoparticles of example 1 as a function of the dye (IR820) concentration in the material; (b) a picture showing photoacoustic imaging of the imaging agent of example 1 comprising IR820-CSQ-Fe nanoparticles;
In FIG. 9, (a) represents T of the imaging agent comprising IR820-CSQ-Fe nanoparticles of example 11A linear plot of the inverse relaxation time as a function of iron concentration; (b) t representing the imaging agent of example 1 comprising IR820-CSQ-Fe nanoparticles2a linear plot of the inverse relaxation time as a function of iron concentration; (c) t representing the imaging agent of example 1 comprising IR820-CSQ-Fe nanoparticles1And T2weighting the imaged picture;
FIGS. 10(a) and (b) are histograms showing the cell viability of MDA-MB-231 cells tested by CCK-8 method after 24 hours of treatment with PBS buffer and CSQ-Fe of example 1 (Fe ion concentration ranging from 0 to 80. mu.g/ml) and an imaging agent comprising IR820-CSQ-Fe nanoparticles (IR820 concentration ranging from 0 to 4. mu.g/ml), respectively;
FIG. 11 is a graph of stability of IR820-CSQ-Fe in water, (a) is a distribution of hydrated particle size of the material IR820-CSQ-Fe after lyophilization and reconstitution, and (b) is a graph of the aqueous solutions of the materials IR820, CSQ-Fe and IR820-CSQ-Fe stored in the dark at room temperature for one day and three months.
Detailed Description
for the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
in the examples, the infrared spectrometer (Spectrum one, Perkin-Elmer, USA) was used as the apparatus for infrared Spectrum detection; the instrument used to measure particle size was a dynamic light scattering instrument (Zetasizer NanoZS); transmission electron microscope (FEI, Tecnai G220S-TWIN, 200kV) with EDAX for particle morphologygenesis 2000XMS energy spectrometer; iron concentration was determined by inductively coupled spectroscopy (PE8000, Perkin Elmer, USA); ultraviolet absorption of the material is performed by using an ultraviolet spectrophotometer (Perkin Elmer Lambda 850); the fluorescence spectrum was measured using a fluorescence spectrophotometer (F-4600, HITACHI, Japan); photoacoustic imaging capability determination using a multispectral small animal in vivo imaging system (MSOT inVision 128, iThera media, germany); t is1And T2The relaxivity was determined using a multi-source magnetic resonance apparatus (BioSpec70/20USR, Bruker).
example 1
This example prepared a multimodal imaging agent using the following steps:
(1) preparation of CSQ-Fe
Dissolving 2g of CSQ with the molecular weight of 100,000Da in 50mL of deionized water, placing the solution in a three-neck flask, introducing nitrogen into a reaction system for 30min, and heating the solution in an oil bath to 102 ℃ to obtain a reaction system A; wherein the molecular formula of the CSQ is as follows:
Wherein n is 32 to 320,
② 0.1459g of FeCl3·6H2O with 0.0715g of FeCl2·4H2dissolving O in 2mL of hydrochloric acid solution with the concentration of 1mol/L to obtain solution B;
Injecting the solution B into the reaction system A in the step (1), and slowly dropwise adding 15mL of concentrated ammonia water until the pH value is 10; refluxing for 40min at 80-120 ℃ to obtain a reaction system C;
fourthly, after the reaction system C in the step (3) is cooled to room temperature, all reaction liquid is moved into a dialysis bag and dialyzed for 72 hours at the temperature of 20-35 ℃ by deionized water, and then the colloid D is obtained.
(2) preparation of IR820-CSQ-Fe
dissolving IR820(15mg, 0.0177mmol) and 6-aminocaproic acid (16.25mg, 0.1239mmol) in 1mL of N, N-Dimethylformamide (DMF), introducing nitrogen for 30min, and heating in oil bath at 85 ℃ to obtain a reaction system E;
② adding 20 μ L Triethylamine (TEA) into the reaction system E, stirring and reacting for 3h in the dark, cooling to room temperature to obtain a reaction system F;
③ adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) (23.75mg, 0.1239mmol) and N-hydroxysuccinimide (NHS) (46.96mg, 0.1239mmol) to the reaction system F to obtain an activated IR820 solution;
Dripping the activated IR820 solution into colloid D containing CSQ-Fe77.8mg, and reacting for 18h in a dark place;
Dialyzing at 20-35 ℃ for 60-75 h, freezing and drying to obtain the imaging agent containing the IR820-CSQ-Fe nanoparticles, and keeping the imaging agent at 4 ℃ in a dark place.
The particles of the imaging agent prepared by the method are spherical through transmission electron microscope observation, the peak value of the hydrated particle size is about 25nm, the mass ratio of iron atoms to chitosan quaternary ammonium salt is 3.1:100 through ICP-OES measurement, and the grafting rate of IR820 is 2.89%.
And (3) performance testing:
When the imaging agent comprising IR820-CSQ-Fe nanoparticles prepared in example 1 and the powder of colloid D obtained in step (1) were subjected to IR spectrum analysis, as shown in FIG. 1, it can be seen that IR820-CSQ-Fe has an IR spectrum of 1500-1700cm relative to CSQ-Fe-1A characteristic peak of the benzene ring appeared, indicating that IR820 had been attached to CSQ-Fe.
When the imaging agent comprising IR820-CSQ-Fe nanoparticles prepared in example 1 and the colloid D, IR820 obtained in step (1) were subjected to UV spectrum analysis, as shown in FIG. 2, it can be seen that an absorption peak at 655nm appeared in the spectrum of IR820-CSQ-Fe relative to CSQ-Fe, whereas the absorption peak in the spectrum of IR820-CSQ-Fe was shifted to a short wavelength relative to IR820, which is due to the extended conjugated structure, indicating that IR820 has been successfully attached to the CSQ-Fe nanoparticles.
The imaging agent comprising IR820-CSQ-Fe nanoparticles prepared in example 1 and the colloid D obtained in step (1) were subjected to hydrated particle size distribution analysis, and the results are shown in FIG. 3. The results show that the particles of the invention have small particle size and good penetrability; the peaks are quite prominent in the figure, and the particle size distribution of the present invention is uniform. Compared with CSQ-Fe, the hydrated particle size peak value of the IR820-CSQ-Fe does not move to a large extent, and the particle size represented by a transmission electron microscope is also similar.
FIG. 4 is a surface potential diagram of the IR820-CSQ-Fe nanoparticle prepared in example 1, and it can be seen that the surface potential of the particle is positive and the peak is prominent, which indicates that the linkage of IR820 does not change the surface potential of CSQ-Fe, and the escape function of the nanoparticle in lysosome after being taken up by the material is not changed.
FIG. 5 is a transmission electron micrograph of CSQ-Fe particles prepared in example 1, which shows that the particles have a small and uniform particle size.
FIG. 6 shows the X-ray energy spectrum of the CSQ-Fe particles prepared in example 1, and the presence of Fe can be seen from the peak in the figure, thus illustrating that the nanoparticles contain Fe.
From FIGS. 1 to 6, it can be seen that IR820-CSQ-Fe has been successfully prepared.
The fluorescence spectrum analysis of the multimode imaging agent nanoparticles prepared in example 1 is carried out, and the result is shown in fig. 7, and it can be seen from the figure that the excitation light of 645nm can obtain the fluorescence with the peak value of 810nm and the fluorescence in the near infrared region, so that the multimode imaging agent nanoparticles can be applied to the fluorescence imaging in vivo.
in fig. 8, (a) shows the photoacoustic imaging signal intensity versus concentration for the multimode imaging agent of example 1, which is seen to be essentially linear over a range of concentrations; (b) indicating that the signal intensity of photoacoustic imaging increases with increasing concentration. The material can therefore be used as an imaging agent for photoacoustic imaging in vivo.
in FIG. 9, (a) shows T of the multimodal imaging agent of example 11A linear plot of the inverse relaxation time as a function of iron concentration; (b) t representing the multimodal imaging agent of example 12a linear plot of the inverse relaxation time as a function of iron concentration; (c) t representing the multimodal imaging agent of example 11and T2The imaged picture is weighted. It can be seen that the T of the particles of the invention1and T2The linear relationship of the inverse relaxation time with the change of the iron concentration is good.
the biocompatibility of the particles prepared in example 1 was then determined by CCK-8 colorimetry, as follows:
The cytotoxicity of the CSQ-Fe nanoparticles and IR820-CSQ-Fe nanoparticles prepared in example 1 was examined by measuring the viability of MDA-MB-231 cells by CCK-8 colorimetry using MDA-MB-231 cells as a model. MDA-MB-231 cells were co-cultured with the particle solutions prepared in example 1 at different concentrations of Fe ions and IR820, respectively (concentrations of 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL, and 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, and 4. mu.g/mL, respectively) at 37 ℃ for 24 hours, and then absorbance at 450nm was measured using CCK-8 kit, and cell viability was calculated based on this value to examine the toxicity of the synthesized material of example 1. As can be seen from fig. 10(a), the first column is a control group, i.e. no Fe ion is added, the cell viability is 100%, compared with the control group, the particles prepared in example 1 have no significant effect on the viability of MDA-MB-231 cells within the experimental concentration range of 5 μ g/mL to 80 μ g/mL, and the cell viability fluctuates slightly within the range of about 100%, indicating no significant cytotoxicity; as can be seen from fig. 10(b), the first column is a control group, i.e., no IR820 ion is added, the cell viability is 100%, compared with the control group, the particles prepared in example 1 have no significant effect on the viability of MDA-MB-231 cells in the experimental concentration range from 0.5 μ g/mL to 4 μ g/mL, and the cell viability fluctuates by a small amount in the range of about 100%, indicating no significant cytotoxicity, which indicates that the particles of the present invention have good biocompatibility in this concentration range.
Example 2
This example prepared a multimodal imaging agent using the following steps:
(1) preparation of CSQ-Fe
dissolving 2g of CSQ with the molecular weight of 10,000Da in 50mL of deionized water, placing the solution in a three-neck flask, introducing nitrogen into a reaction system for 30min, and heating the solution in an oil bath to 102 ℃ to obtain a reaction system A; wherein the molecular formula of the CSQ is as follows:
Wherein n is 32 to 320,
② 0.1459g of FeCl3·6H2O with 0.0715g of FeCl2·4H2Dissolving O in 2mL of hydrochloric acid solution with the concentration of 1M to obtain solution B;
injecting the solution B into the reaction system A in the step (1), and slowly dropwise adding 15mL of concentrated ammonia water until the pH value is 10; refluxing at high temperature for 40min to obtain a reaction system C;
Fourthly, after the reaction system C in the step (3) is cooled to room temperature, all reaction liquid is moved into a dialysis bag and dialyzed for 72 hours at the temperature of 20-35 ℃ by deionized water, and then the colloid D is obtained.
(2) Preparation of IR820-CSQ-Fe
Dissolving IR820(15mg, 0.0177mmol) and 6-aminocaproic acid (16.25mg, 0.1239mmol) in 1mLN, N-Dimethylformamide (DMF), introducing nitrogen for 30min, and heating in oil bath at 85 ℃ to obtain a reaction system E;
② adding 20 μ L Triethylamine (TEA) into the reaction system E, stirring and reacting for 3h in the dark, cooling to room temperature to obtain a reaction system F;
③ adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) (23.75mg, 0.1239mmol) and N-hydroxysuccinimide (NHS) (46.96mg, 0.1239mmol) to the reaction system F to obtain an activated IR820 solution;
Dripping the activated IR820 solution into colloid D containing CSQ-Fe77.8mg, and reacting for 18h in a dark place;
Dialyzing at 20-35 ℃ for 60-75 h, freezing and drying to obtain the imaging agent containing the IR820-CSQ-Fe nanoparticles, and keeping the imaging agent at 4 ℃ in a dark place.
The imaging agent prepared in example 2 and comprising the IR820-CSQ-Fe nanoparticles is spherical through transmission electron microscope observation, the peak value of the hydrated particle size is about 30nm, the mass ratio of iron atoms to chitosan quaternary ammonium salt is 5.2:100 through ICP-OES measurement, and the grafting ratio of IR820 is 8.1%.
example 3
This example prepared a multimodal imaging agent using the following steps:
(1) Preparation of CSQ-Fe
Dissolving 2g of CSQ with the molecular weight of 100,000Da in 50mL of deionized water, placing the solution in a three-neck flask, introducing nitrogen into a reaction system for 30min, and heating the solution in an oil bath to 102 ℃ to obtain a reaction system A; wherein the molecular formula of the CSQ is as follows:
wherein n is 32 to 320,
② 0.1459g of FeCl3·6H2o with 0.0715g of FeCl2·4H2Dissolving O in 2mL of hydrochloric acid solution with the concentration of 1M to obtain solution B;
injecting the solution B into the reaction system A in the step (1), and slowly dropwise adding 15mL of concentrated ammonia water until the pH value is 10; refluxing at high temperature for 40min to obtain a reaction system C;
fourthly, after the reaction system C in the step (3) is cooled to room temperature, all reaction liquid is moved into a dialysis bag and dialyzed for 72 hours at the temperature of 20-35 ℃ by deionized water, and then the colloid D is obtained.
(2) Preparation of IR820-CSQ-Fe
Dissolving IR820(5mg, 0.0067mmol) and 6-aminocaproic acid 16.25mg, 0.1239mmol in 1mLN, N-Dimethylformamide (DMF), introducing nitrogen for 30min, heating in oil bath at 85 ℃ to obtain a reaction system E;
② adding 20 μ L Triethylamine (TEA) into the reaction system E, stirring and reacting for 3h in the dark, cooling to room temperature to obtain a reaction system F;
③ adding 23.75mg and 0.1239mmol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 46.96mg and 0.1239mmol of N-hydroxysuccinimide (NHS) into the reaction system F to obtain an activated IR820 solution;
dripping the activated IR820 solution into colloid D containing CSQ-Fe77.8mg, and reacting for 18h in a dark place;
And fifthly, dialyzing at 20-35 ℃ for 60-75 h, freezing and drying to obtain the imaging agent, and storing at 4 ℃ in a dark place.
The peak value of the hydrated particle size of the imaging agent containing the IR820-CSQ-Fe nano particles prepared in example 3 is about 90nm through transmission electron microscope observation, the mass ratio of iron atoms to chitosan quaternary ammonium salt is 3.1:100 through ICP-OES measurement, and the grafting rate of IR820 is 1%.
Example 4
this example prepared a multimodal imaging agent using the following steps:
(1) Preparation of CSQ-Fe
Dissolving 2g of CSQ with the molecular weight of 10,000Da in 50mL of deionized water, placing the solution in a three-neck flask, introducing nitrogen into a reaction system for 30min, and heating the solution in an oil bath to 80 ℃ to obtain a reaction system A; wherein the molecular formula of the CSQ is as follows:
Wherein n is 32 to 320,
② 0.1459g of FeCl3·6H2o with 0.0715g of FeCl2·4H2Dissolving O in 2mL of hydrochloric acid solution with the concentration of 1M to obtain solution B;
injecting the solution B into the reaction system A in the step (1), and slowly dropwise adding 15mL of concentrated ammonia water until the pH value is 10; refluxing at high temperature for 40min to obtain a reaction system C;
Fourthly, after the reaction system C in the step (3) is cooled to room temperature, all reaction liquid is moved into a dialysis bag and dialyzed for 72 hours at the temperature of 20-35 ℃ by deionized water, and then the colloid D is obtained.
(2) Preparation of IR820-CSQ-Fe
Dissolving IR820(15mg, 0.0177mmol) and 6-aminocaproic acid (16.25mg, 0.1239mmol) in 1mLN, N-Dimethylformamide (DMF), introducing nitrogen for 30min, and heating in oil bath at 85 ℃ to obtain a reaction system E;
② adding 20 μ L Triethylamine (TEA) into the reaction system E, stirring and reacting for 3h in the dark, cooling to room temperature to obtain a reaction system F;
③ adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) (23.75mg, 0.1239mmol) and N-hydroxysuccinimide (NHS) (46.96mg, 0.1239mmol) to the reaction system F to obtain an activated IR820 solution;
Dripping the activated IR820 solution into colloid D containing CSQ-Fe77.8mg, and reacting for 18h in a dark place;
And fifthly, dialyzing at 20-35 ℃ for 60-75 h, freezing and drying to obtain the imaging agent, and storing at 4 ℃ in a dark place.
the imaging agent prepared in example 4 and comprising IR820-CSQ-Fe nanoparticles is observed by a transmission electron microscope, the peak value of the hydrated particle size is about 45nm, the mass ratio of iron atoms to chitosan quaternary ammonium salt measured by ICP-OES is 5.2:100, and the grafting rate of IR820 is 6.2%.
example 5
The only difference from example 1 is: in the step (2), 6-aminocaproic acid of the first step is replaced by 6-aminobutyric acid in an equimolar way.
The imaging agent comprising IR820-CSQ-Fe nanoparticles prepared in example 4 was observed by transmission electron microscopy as shown in FIG. 5 (b).
Example 7
a portion of IR820-CSQ-Fe was removed and lyophilized, then 10mg of the material was reconstituted with 3mL of physiological saline and the hydrated particle size was measured using a laser particle sizer, as shown in FIG. 11a, in the same manner as in example 1. As can be seen from fig. 11a, the resulting hydrated particle size after nanomaterial reconstitution is almost indistinguishable from the initial assay (fig. 3). Therefore, the material can be stored for a long time after being freeze-dried, and the defects that some inorganic materials are unstable in water and cannot be redissolved after being freeze-dried are overcome.
and FIG. 11b is a photograph of an aqueous solution of the materials IR820-CSQ-Fe and CSQ-Fe stored in the dark at room temperature for three months, from which the good stability of these two materials in water can be seen.
The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (35)
1. a composite imaging agent integrating nuclear magnetic resonance imaging, fluorescence imaging and photoacoustic imaging is characterized in that the composite imaging agent comprises IR820-CSQ-Fe nanoparticles, and the IR820-CSQ-Fe nanoparticles are ferroferric oxide coated with the following structures:
A-X-B；
Wherein A is chitosan quaternary ammonium salt CSQ with a structure of formula (I):
wherein n is an integer of 32-320;
B is a novel indocyanine green IR820 having the structure of formula (II):
X is 6-aminocaproic acid;
The particle size of the IR820-CSQ-Fe nano particles is 10-30 nm;
the hydration particle size of the IR820-CSQ-Fe nano particles is 20-100 nm;
The mass ratio of iron atoms in the IR820-CSQ-Fe nanoparticles to the mass ratio of IR820 to CSQ is (3.1-5.2): 1-8.1): 100;
The composite imaging agent is prepared by adopting the following method, and the preparation method comprises the following steps:
(1) Preparation of CSQ-Fe:
Dissolving CSQ in deionized water, continuously introducing protective gas, heating, and preserving heat to obtain a reaction system A;
dissolving trivalent ferric salt and divalent ferric salt in strong acid solution to obtain solution B;
Thirdly, injecting the solution B into the reaction system A, adjusting the pH value to be alkaline, and refluxing to obtain a reaction system C;
cooling the reaction system C to room temperature, and dialyzing to obtain a colloid D, namely CSQ-Fe;
(2) Preparation of IR820-CSQ-Fe nanoparticles
dissolving IR820 and 6-aminocaproic acid in an organic solvent, continuously introducing inert gas, heating, and keeping the temperature to obtain a reaction system E;
adding organic amine into the reaction system E, stirring and reacting in the dark, and cooling to room temperature to obtain a reaction system F;
Contacting carbodiimide condensing agent and acylation activating agent with the same molar amount as 6-aminocaproic acid with the reaction system F to obtain activated IR820 solution;
dripping the activated IR820 solution into the colloid D, and reacting at room temperature in a dark place;
Dialyzing and freeze-drying to obtain the imaging agent containing the IR820-CSQ-Fe nano particles, and storing at 4 ℃ in a dark place.
2. A method of preparing the composite imaging agent of claim 1, comprising the steps of:
(1) preparation of CSQ-Fe:
Dissolving CSQ in deionized water, continuously introducing protective gas, heating, and preserving heat to obtain a reaction system A;
dissolving trivalent ferric salt and divalent ferric salt in strong acid solution to obtain solution B;
Thirdly, injecting the solution B into the reaction system A, adjusting the pH value to be alkaline, and refluxing to obtain a reaction system C;
Cooling the reaction system C to room temperature, and dialyzing to obtain a colloid D, namely CSQ-Fe;
(2) Preparation of IR820-CSQ-Fe nanoparticles
Dissolving IR820 and 6-aminocaproic acid in an organic solvent, continuously introducing inert gas, heating, and keeping the temperature to obtain a reaction system E;
adding organic amine into the reaction system E, stirring and reacting in the dark, and cooling to room temperature to obtain a reaction system F;
Contacting carbodiimide condensing agent and acylation activating agent with the same molar amount as 6-aminocaproic acid with the reaction system F to obtain activated IR820 solution;
Dripping the activated IR820 solution into the colloid D, and reacting at room temperature in a dark place;
dialyzing and freeze-drying to obtain the imaging agent containing the IR820-CSQ-Fe nanoparticles, and keeping the imaging agent at 4 ℃ in a dark place;
the mass ratio of the iron element, the IR820 and the chitosan quaternary ammonium salt CSQ is (3.1-5.2): (1-8.1): 100.
3. The method for preparing the composite imaging agent according to claim 2, wherein the concentration of CSQ in the reaction system A in step (1) is 30 to 60 g/L.
4. the method for producing a composite imaging agent according to claim 2, wherein (1) the protective gas is any 1 or a combination of at least 2 of nitrogen and/or an inert gas.
5. the method of producing a composite imaging agent according to claim 2, wherein (1) the protective gas is nitrogen and/or argon.
6. The method for preparing a composite imaging agent according to claim 2, wherein (1) the incubation temperature is 80 to 120 ℃.
7. The method for preparing a composite imaging agent according to claim 2, wherein in the step (1), the incubation time is 20-50 min.
8. The method for preparing the composite imaging agent according to claim 2, wherein the molar ratio of the ferric salt to the ferrous salt in step (1) is 2 (1-2).
9. the method of claim 2, wherein the trivalent iron salt is FeCl in the step (1)3·6H2O。
10. The method of preparing a composite imaging agent according to claim 2, wherein the ferrous salt is FeCl in step (1)2·4H2O or FeSO4·7H2O。
11. The method of preparing the composite image forming agent according to claim 2, wherein the concentration of the strong acid in the strong acid solution in the step (1) is 0.5 to 3moL/L, the concentration of the ferric ion is 0.4 to 1g/L, and the concentration of the ferrous ion is 0.2 to 1 g/L.
12. The method of claim 2, wherein the agent for adjusting pH in step (1) is an alkali.
13. the method of claim 2, wherein the agent for adjusting pH in step (1) is ammonia.
14. The method for preparing the composite imaging agent according to claim 2, wherein the reagent for adjusting the pH in the step (1) is ammonia water with a mass fraction of 25-28%.
15. the method of claim 2, wherein the step (1) of adjusting the pH to alkaline is performed to adjust the pH to 8 to 11.
16. The method for preparing the composite imaging agent according to claim 2, wherein the time for the reflux in step (1) is 40-120 min, and the temperature for the reflux is 80-120 ℃.
17. The method of preparing a composite imaging agent according to claim 2, wherein the dialysis in step (1) is dialysis with deionized water.
18. The method for preparing a composite imaging agent according to claim 2, wherein the dialysis temperature in step (1) is 20 to 35 ℃.
19. The method for preparing a composite imaging agent according to claim 2, wherein the dialysis time in step (1) is 60 to 75 hours.
20. the process for producing an imaging agent according to claim 2, wherein in the step (2), the molar ratio of IR820 to 6-aminocaproic acid is 1 (5 to 10).
21. The method for preparing a composite imaging agent according to claim 2, wherein (2) the organic solvent is selected from the group consisting of N, N-dimethylformamide and/or dimethylsulfoxide; the dosage of the organic solvent is 1-10 mL of the organic solvent used per 15mg of IR 820.
22. The method for producing a composite imaging agent according to claim 2, wherein (2) the inert gas is any 1 or a combination of at least 2 of nitrogen and/or an inert gas.
23. the method for producing a composite image forming agent according to claim 2, wherein (2) the inert gas is nitrogen and/or argon.
24. the method for preparing a composite imaging agent according to claim 2, wherein (2) the incubation temperature is 80 to 90 ℃.
25. The method for producing a composite image forming agent according to claim 2, wherein (2) the incubation temperature is 85 ℃.
26. The method for preparing a composite imaging agent according to claim 2, wherein in the step (2), the incubation time is 20-50 min.
27. The method of preparing a composite imaging agent according to claim 2, wherein the organic amine in step (2) is selected from triethylamine and/or N, N-diisopropylethylamine; in the reaction system F, the molar ratio of the organic amine to the 6-aminocaproic acid is (1-5): 1.
28. The method for preparing a composite imaging agent according to claim 2, wherein the stirring reaction time in the step (2) is 2 to 4 hours.
29. the method of claim 2, wherein the stirring reaction time in step (2) is 3 hours.
30. The method for producing a composite imaging agent according to claim 2, wherein the carbodiimide condensing agent in step (2) is 1-ethyl- (3-dimethylaminopropyl) carbodiimide and/or N, N-dicyclohexylcarbodiimide.
31. the method of claim 2, wherein step (2) the acylating activator is N-hydroxysuccinimide and/or 1-hydroxybenzotriazole.
32. The method for preparing a composite imaging agent according to claim 2, wherein the molar ratio of the IR820 to CSQ in the colloid D in the step (2) is 1:20 to 200; the reaction time is 16-24h without light.
33. The method of claim 2, wherein the dialysis in step (2) is performed with deionized water.
34. the method for preparing a composite imaging agent according to claim 2, wherein the dialysis temperature in step (2) is 20 to 35 ℃.
35. The method for preparing a composite imaging agent according to claim 2, wherein the dialysis time in step (2) is 60 to 75 hours.
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